Molecular Cloning, Expression And Sequence Analysis On Genes Related To Nodal Signaling Pathway In Flatfish

In this study, the most stable genes for Real time RT-PCR were selected throughthe expression analysis of housekeeping genes. Real time RT-PCR is referred to as themost sensitive method for the detection of low abundance mRNA. To date, noreference genes have been validated for development gene expression studies in Half-smooth tongue sole. Eight housekeeping genes (18S rRNA, TUBA, B2M, ACTB,EF1A, GAPDH, RPL17and UBCE) were tested during development stages, tissuesand chemical exposure using geNorm and Bestkeepper software. The top twopositions of reference genes, GAPDH and B2M, were sifted out as the most suitableones for samples of different development stages by geNorm. GAPDH/18S wasranked as the best internal reference genes and B2M was fall down to third byBestkeeper.18S and RPL17for samples of different tissue types, as consistent resultsof two software; B2M and TUBA for samples of drug treatment by geNorm andUBCE/TUBA was stand out by Bestkeeper. Across developmental stages and tissuetypes,18s and GAPDH were found to be most stable reference genes. These resultscan be used for future gene expression studies in half smooth tongue sole.lefty and pitx2sequences were cloned from Half-smooth tongue sole（Cynoglossus semilaevis） by homology cloning approach and RACE PCR. Theopen reading frame of lefty was972bp, encoding324amino acids. And the length ofpitx2was939bp for open reading frame, encoding313amino acids. The proteinencoded by pitx2presented extremely conservative in the area of the Homeobox andOAR domains. Both the expression of lefty and pitx2were appeared maternalphenomenon. lefty showed transient expression, mainly concentrating in theembryonic development stages. The expression of pitx2, however, was detected injuvenile fish with a slightly decreasing dose, in addition to the embryonic stages. Using homology-based cloning combined with a gDNA library of methods, fulllength cDNA sequences of dnah9were obtained from the Japanese flounder(Paralichthys olivaceus). The open reading frame of cDNA was13611bp, and therewas384bp in3’UTR region, encoding4573amino acids. There were two copies ingenomic DNA for the tandem arrangement: the first copy was62632bp with100exons. Second copy was128387bp with80exons. dnah9showed maternalexpression phenomenon, Its expression reduced gradually after fertilization, andrebounded after the blastula stage, and reached its peak period in15somites. Embryoshowed that the expression mainly was in several regions of the head: diencephalon,hindbrain and medulla oblongata by in situ hybridization.